Neurons and endocrine cells release signaling molecules by Ca2+?triggered exocytosis. Ca2+ enters a nerve terminal or endocrine cell, binds to a Ca2+ sensor protein on the vesicle membrane, and triggers the fusion of the vesicle membrane with the plasma membrane to expel some or all of the vesicle content into the extracellular space. To explore the mechanisms of this process our research focuses on the fusion pore, the initial aqueous passage between the vesicle interior and the outside of a cell. Studies of the fusion pore have given us valuable insights into the roles of specific molecules in the regulation of membrane fusion. The previous funding cycle saw progress in the identification of the vesicle protein synaptobrevin 2 as a fusion pore?forming protein in endocrine cells. This work revealed new unanticipated details in the structure of the fusion pore. We also showed that the transmembrane domain of this protein alters fusion pore stability by perturbing lipid bilayers. These findings raise new questions which we will attempt to answer by performing amperometry and capacitance recordings along with recordings of unitary synaptic current to obtain sensitive measures of fusion pore permeability and stability. We will build on insights from studies of endocrine fusion pores to test the role of the transmembrane domains of syntaxin and synaptobrevin in synaptic vesicle fusion pores. We will test the role of the vesicle protein synaptophysin in endocrine fusion pores by assaying flux and lipid perturbation. We will test the role of contacts between linkers and transmembrane domains of syntaxin and synaptobrevin in providing an energetic driving force for fusion pore expansion. These experiments will establish the specific functions of proteins associated with exocytosis. We will determine how these proteins contribute to the formation of specific structural intermediates of fusion, and how these proteins deform and remodel lipid bilayers to carry out these functions.